Despite considerable scientific and industrial efforts, there are no drugs available for an efficient treatment of alcoholism. This is partially explained by the fact, that alcohol, unlike most psychoactive drugs, does not produce its actions via a cognate receptor system. Instead, alcohol modulates directly or indirectly a variety of signaling systems including the CNS opioid signaling system. Alcohol-opioid interactions are complex and reflected at the systems, synaptic, cellular and molecular levels. However, the mechanisms mediating alcohol-opioid interactions have yet to be fully resolved. Recent advances in molecular imaging now offer new approaches to study alcohol-opioid interactions at the level of opioid receptor, and especially interactions that affect receptor dynamics, which plays a central role in determining receptor availability and function. To this end, we are currently involved in the development of Fluorescence Correlation Spectroscopy (FCS) integrated with Confocal Laser Scanning Microscopy (CLSM) for nondestructive observation of molecular interactions in living cells in real time with ultimate single-molecule sensitivity. We propose to utilize this system and parallel functional analysis to develop approaches for the investigation of alcohol (i.e., ethanol) effects on cellular dynamics of opioid receptors and the functional consequence of these interactions. Based on our preliminary studies we propose that ethanol-induced changes in opioid receptor dynamics play an important role in alcohol-opioid interactions in the CNS and hypothesize that such interactions lead to functional changes in neuronal physiology. We also hypothesize that an important action of opioid receptor antagonists known to have therapeutic potential for alcoholism such as naltrexone is to block the action of ethanol on opioid receptor dynamics. To test these hypotheses, we propose 3 Specific Aims: (1) Develop a cellular model to study interactions between alcohol and the opioid signaling system at the level of opioid receptor dynamics using FCS/CLSM. These studies will employ PC12 cells transfected with tagged or untagged opioid receptors. (2) Identify the consequences of alcohol induced changes in opioid receptor dynamics to neurophysiology using electrophysiological recordings of neuronal properties, and (3) extend studies of alcohol regulation of opioid receptor dynamics to native neurons and neuronal circuits where investigations of ethanol/opioid interactions can be pursued in cells and neuronal circuits more reflective of the in vivo conditions. These studies will employ primary cultures of mouse hippocampus obtained from opioid receptor knockout mice. The cultured cells will be transfected with tagged or untagged opioid receptors. The proposed studies represents a novel and state of the art approach to an understanding of mechanisms involved in ethanol-opioid interactions including mechanisms that may be central to the development of alcohol dependence. Project Narrative Alcohol is one of the most widely abused psychoactive substances in the world. Several lines of evidence suggest that the brain opioid signaling system mediates many of the important behavioral effects alcohol associated with alcohol abused. The proposed studies will investigate actions of alcohol that occur at the level of the opioid receptor level to gain an understanding of mechanisms that mediate alcohol-opioid interactions.